This invention relates to new varieties of plants and the production thereof, and, more particularly, to new varieties of plants having enhanced performance and a method of producing same.
Plant breeders are continually developing new plant varieties in which desirable characteristics and plant performance are optimized. Plant performance is a reflection of the sum total of many factors, including yield or productivity, ecological fitness, appearance, vigor, resistance to weed invasion, recovery from injury, persistence, and density, and can be enhanced by improving pest resistance and tolerance of herbicides, defoliation, heat, and drought.
Resistance to insect predation is an important factor in any given plant's performance. Consequently, plant breeders constantly seek to upgrade the insect resistance of important plant varieties. However, as soon as anew variety of insect-resistant plant is developed, usually after years of painstaking breeding programs, insects may sooner or later evolve that are able to feed, without adverse effect, on the once insect-resistant plant. Thus, the ultimate grower of the new plant variety is faced with a number of alternatives. He can either await further development of a new variety of pest-resistant plant, or turn either to chemical pesticides or biological pest control.
Generally, chemical pesticides are unduly expensive, and quite frequently they have an objectionable environmental impact.
An alternative to the use of chemical pesticides is biological pest control. Perhaps the best known use of biological pest control is the well-publicized case of the screwworm fly. There, the discovery that screwworm flies mated only once led to the method whereby large numbers of laboratory-bred male flies were sterilized by X-ray irradiation. By subsequently releasing these sterile males, the females with which they mated could lay only infertile eggs. Thus, by exploiting the known mating habits of a particular insect pest, its numbers were effectively curtailed. Another example of biological pest control includes the use of insect pathogens, such as certain lethal or debilitating insect viruses. Because these viruses are generally host-specific, the targeted insect pest can be readily controlled without harming beneficial species.
The advantages of biological control of insect pests are several. First, biological controls are generally self-limiting; once numbers of the target species are reduced, so too are the biological controls. Second, biological pest controls are usually host-specific and do not attack desirable species. Finally, and perhaps most importantly, biological pest controls are normally environmentally compatible, unlike chemical pesticides which may persist in the environment and kill indiscriminately.
A new biological pest control has recently been recognized. Certain plants host symbiotic endophytic fungi which confer, among other things, an enhanced resistance to insect predation on the host plant. For example, in perennial rye-grasses, a positive association has been demonstrated between the presence of an endophytic fungus (literally, a fungus living within its plant host) and resistance of the plant to attack by some of the most prevalent insect infestations encountered in the field--i.e. the sod webworm, the bluegrass billbug, the Argentine stem weevil, the Southern armyworm, and the chinch bug.
In particular, perennial ryegrasses of the genus Lolium hosting an endophytic fungus are highly resistant to feeding of the larval stages of the Lepidopteran sod webworms of the genus Crambus. Commonly found Crambus species include C. mutabilis, C. teterrellus, C. trisectus, and C. vulgivagellus. Plants lacking the endophytic fungus can sustain substantial injury from feeding Crambus larvae. (C. R. Funk et al., 1983, "Implications of Endophytic Fungi in Breeding for Insect Resistance," Proc. Forage and Turfgrass Endophyte Workshop, pp. 67-75, and C. R. Funk et al., 1983, "An Endophytic Fungus and Resistance to Sod Webworms: Association in Lolium perenne L.," Bio/Technology 1(2):189, each incorporated herein by reference.) Resistance in ryegrasses hosting this fungus to feeding of the larval stages of the Coleopteran bluegrass billbug (Sphenophorus spp.) has also been observed. (Funk et al., 1983, Proc. Forage and Turfgrass Endophyte Workshop, pp. 67-68.) Finally, in ryegrasses hosting an endophytic fungus we have observed resistance to feeding by the Hemipteran chinch bug (Blissus spp.), and others have observed resistance in ryegrasses hosting endophytic fungus to the Coleopteran Argentine stem weevil (Listronotus spp.). This endophyte-enhanced insect resistance in ryegrasses to three different orders of very prevalent chewing insects (Lepidoptera, Coleoptera, and Hemiptera) provided us with a broad-based mechanism for developing new plants having enhanced performance including resistance to these insects.
The exact mechanism of this enhanced resistance to insect predation has not as yet been identified, although it is suspected that such resistance could involve the generation of chemicals toxic to insects which may be present in plants containing the endophytic fungi. These chemicals might be produced by the endophytic fungus or by the host plants themselves in response to the invading fungus. The latter mechanism may mediate a generalized resistance to insects feeding on plant parts having the highest concentrations of endophytic fungi or their associated toxins.
By way of example, the association of sod webworm resistance with the presence of an endophytic fungus in several varieties, selections, and single-plant progenies of perennial ryegrasses was tested in ryegrass turf trials at Adelphia, N.J. in 1980. Resistance was expressed both as a lack of larval feeding and as a nearly complete absence of larvae from the soil beneath resistant ryegrasses. Results of that test are summarized in Table 1, from C. R. Funk et al., 1983, Bio/Technology 1(2):189 at 190. Perennial ryegrass entries containing a high percentage of the endophytic fungus showed resistance to predation by the sod webworm. Ryegrasses susceptible to sod webworm predation contained zero or low levels of the endophyte.
Maternal transmission (seed produced from a mother plant high in endophyte) of insect resistance was dramatically evident in the Adelphia test. All open-pollinated, single-plant progenies descending from lines without endophyte produced susceptible progenies. Progenies produced from seed of each of the single plants could be categorized as either resistant or susceptible; no seedlings were rated as having intermediate susceptibility. Essentially all plants produced by seed harvested from a given single plant were either resistant or susceptible depending on the presence or absence of the endophyte in the mother plant, an unexpected result because perennial ryegrass is a highly heterozygous, cross-pollinated species.
TABLE 1 ______________________________________ Association of sod webworm resistance with presence of an endophytic fungus in cultivars, selections, and single-plant progenies of perennial ryegrass. Endophyte level in seed Percent.sup.a Microscopic ELISA ANALYSIS green examination Endophyte Entry turfgrass %.sup.b Presence.sup.c %.sup.d ______________________________________ Ryegrasses rated as resistant to sod webworms.sup.e 1. 79-132 95 93 + 87 2. 79-140 90 96 + 100 3. 79-153 98 97 + 90 4. 79-376 98 97 + 90 5. Pennant 87 90 + 87 6. SWRC-1 88 + 87 7. 79-130 98 100 8. 79-141 96 97 9. 79-157 90 100 10. 79-249 95 100 11. 79-361 95 100 12. GT-II 96 98 Ryegrasses rated as susceptible to sod webworms.sup.e 13. 79-135 15 0 - 14. 79-137 15 0 - 15. 79-164 10 0 .+-. 0 16. 79-244 30 0 - 17. 79-268 20 0 .+-. 0 18. 79-136 30 0 19. 79-146 12 0 20. 79-159 13 0 21. 79-269 20 0 22. 79-389 30 0 23. Gator 14 2 24. Yorktown 16 0 .+-. 0 II 25. Diplomat 20 0 - ______________________________________ .sup.a % green turfgrass is an indication of absence of injury by sod webworm larvae. .sup.b % infection based on an analysis of 3050 individual seeds per entry. .sup.c ELISA analysis based on 5 lots of 10 seeds each (50 seeds total pe entry). Minus (-): all 5 seed lots negative. Plus-minus (.+-.): very weak ELISA reactions in 1 or 2 of the 5 seed lots Plus (+): all 5 seed lots strongly positive. .sup.d % infection based on an analysis of 30 individual seeds per entry. .sup.e Resistant ryegrasses had an average of 0.74 sod webworm larvae per 0.1 m.sup.2, whereas susceptible ryegrasses had from 8.0 to 13.7 sod webworm larvae per 0.1 m.sup.2. The observation of maternal transmissio of sod webworm resistance is illustrative of enhanced performance due to endophyte-enhanced pest resistance.
In addition to the observed resistance to predation by insects, plants hosting the endophytic fungus have displayed a certain enhanced performance which includes increased yield or productivity, improved ecological fitness, a more attractive appearance, increased vigor, reduced weed invasion, more rapid recovery from injury, improved persistence, increased density, and apparently greater stress tolerance. For example, in turf trials of tall fescue and perennial ryegrass varieties and single-plant progenies established during the late summer of 1976 at North Brunswick, N.J., those varieties containing a high level of endophytic fungus showed dramatically improved performance after seven years. Species tested included tall fescue (Festuca arundinacea) and ryegrass (Lolium perenne). These plants were more persistent, showed reduced crabgrass invasion, produced a higher yield, had greater vigor, and displayed an improved appearance. Much of this improved performance of these fungal-endophyte-hosting plants appears to be associated with improved stress tolerance, such as tolerance of herbicides, heat, drought, and defoliation. Similar enhanced performance, including resistance to the billbug and the chinch bug, has been observed for hard fescue (Festuca longifolia) and for chewings fescue (Festuca rubra).
The particular endophytic fungus involved in the above-described insect resistance and enhanced performance in ryegrass has been provisionally designated the Lolium endophyte. A similar or identical endophytic fungus present within tall fescue (Festuca arundinacea Schreb.) has been identified as Epichloe typhina (Fr.) Tul. and was recently renamed Acremonium coenophialum Morgan-Jones and Gams.
The life cycles of endophytic fungi have been studied in detail. (C. W. Bacon et al., 1983, "Biology of the Endophyte of Fescue: Ultrastructural Analysis and Physiological Relationships," Proc. of the Forage and Turfgrass Endophyte Workshop, pp. 19-28.) The fungus begins within the seed of the host plant, adjacent to the aleurone layer. When the seed germinates, the fungus spreads into the endosperm, from which the developing embryo derives nutrient, and subsequently into the embryo or developing seedling. Apparently, as the seedling develops strengthening tissue and air spaces, the fungus is able to grow between the plant's cells, i.e. interstitially. In the mature plant, the fungus grows into the rhizomes, leaf seed tissue, flower stem, and seeds, but avoids penetration into the roots.
As a prelude to the invasion of the fungus into its host's developing seed, the fungus concentrates its mycelia in the flower stem. As the seed develops, the fungus grows into the seed adjacent to the aleurone layer, initially avoiding the embryo. Upon germination, invasion of the embryo begins, and the fungus life cycle continues as just described.